No abstract

The rapid development of CRISPR–Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3′UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR–Cas orthologue, for which a potent anti-CRISPR protein is known.

Apoptosis occurs through a tightly regulated cascade of caspase activation. In the context of extrinsic apoptosis, caspase-8 is activated by dimerization inside a death receptor complex, cleaved by auto-proteolysis and subsequently released into the cytosol. This fully processed form of caspase-8 is thought to cleave its substrates BID and caspase-3. To test if the release is required for substrate cleavage, we developed a novel approach based on localization probes to quantitatively characterize the spatial-temporal activity of caspases in living single cells. Our study reveals that caspase-8 is significantly more active at the plasma membrane than within the cytosol upon CD95 activation. This differential activity is controlled by the cleavage of caspase-8 prodomain. As a consequence, targeting of caspase-8 substrates to the plasma membrane can significantly accelerate cell death. Subcellular compartmentalization of caspase-8 activity may serve to restrict enzymatic activity before mitochondrial pathway activation and offers new possibilities to interfere with apoptotic sensitivity of the cells. Apoptosis is coordinated by the activity of initiator and effector caspases.1-4 While effector caspases are dimeric zymogens that become activated by cleavage, initiator caspases are normally expressed as monomeric zymogens and their activity is initiated by dimerization in a multimeric complex. 5,6 The initiator caspase procaspase-8 dimerizes in the death-inducing signaling complex (DISC) formed around activated death receptors. 7 In the context of CD95, the DISC contains clustered receptors bound to the adaptor protein FADD. FADD can recruit several proteins, 8,9 including procaspase-8 and -10 through their prodomain. In type I cells, active caspase-8/10 directly cleaves and activates effector caspase-3/-7, thus inducing apoptosis. In type II cells, this activation is blocked by XIAP, but cleavage of BID by active caspase-8/10 induces mitochondrial outer membrane permeabilization (MOMP), followed by initiator caspase-9 activation and release of XIAP-inhibitor SMAC, leading to massive caspase-3/7 activity. 10,11 Biochemical and structural studies showed that dimerization but also cleavage in the catalytic subunit of caspase-8 is required for efficient cleavage of caspase-3 and BID and for apoptosis.12-14 Supporting this, the non-cleavable D387A caspase-8 mutant compromises apoptosis in mice. 15Although caspase-8 dimerization generates some activity, cleavage in the catalytic units likely stabilizes the active form and increases activity. 13,16 Caspase-8 is also cleaved between the prodomain and the catalytic unit of the enzyme, at D210 and D216, 17,18 and subsequently released from the DISC. Active caspase-8 can be detected on the plasma membrane, before release, with fluorescent inhibitors. 19 In contrast, by designing a procaspase-8 artificially dimerized on the plasma membrane, Martin et al.18 suggested that the cytosolic release is necessary to cleave caspase-3 and BID. Other studies proposed that fully processed ...